Project 3 - Antibiotic resistance and metabolic pathways in Chlamydia spp This project will focus on key biosynthetic and metabolic functions of Chlamydia as they impact on drug resistance and growth and survival in the host. It consists of three aims: Specific Aim 1 - To model the emergence of resistance to the drugs of choice for the treatment of C. trachomatis infections. We will measure the fitness of azithromycin resistant (AZM*^) mutants in vitro and in vivo. We will then screen for compensatory mutants that arise in vivo and characterize these mutants using whole genome sequencing technology. We will also select for spontaneous tetracycline resistant (Tc*^) mutants of C. caviae (GPIC) in a natural infection model in guinea pigs. The mutants that arise in the natural infection setting will be analyzed for growth characteristics such as growth in tissue culture and competition experiments in vivo and in vitro in the absence of antibiotic. A complement to the animal model studies will be a survey for drug resistant Isolates the clinical setting among treatment failures (Clinical Core). Specific Aim 2 - Biosynthetic and metabolic pathway hole filling - characterize the biochemical and genetic components of essential biosynthetic and metabolic pathways of Chlamydia spp. This aim will link the antibiotic resistance aim of the project with biosynthetic pathway hole filling by focusing on the peptidoglycan paradox and transpeptidatlon. We will determine the molecular basis of sensitivity of C. trachomatis to p-lactam antibiotics (amoxicillin/penicillin). The metabolic pathway hole filling component of this aim will address three essential growth factors for which Chlamydia has Incomplete pathways for synthesis: folate, llpolc acid and NAD. In each case we will examine the possibility that Chlamydia express enzymes for de novo synthesis of these factors or whether they express novel transport systems for their uptake from the host cytosol. Genetic and biochemical approaches will be used in surrogate bacterial systems due to the unavailability of genetic techniques for creating mutants in Chlamydia. Specific Aim 3 - Transcriptome mapping of GPIC gene expression profile during infection in different anatomical sites (ocular vs. genital) in guinea pig. Guinea pigs will be infected with two different clonal strains of GPIC at ocular and genital sites and microarrays will be used to determine niche specific gene expression profiles. The expression profiles to be compared are: 1) ocular site vs. genital site; 2) genitally infected males vs. genitally infected females. Highly differentially expressed genes/pathways will be chosen for detailed in vitro analysis (metabdomics) with the goal of exploring pathway/enzyme as targets for inhibitors and eventually new therapeutics.